Differential Gene Expression in Porcine Lung Compartments after Experimental Infection with Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae (Mhyo) is the causative agent of porcine enzootic pneumonia (EP), as well as one of the main pathogens involved in the porcine respiratory disease complex. The host-pathogen interaction between Mhyo and infected pigs is complex and not completely understood; however, improving the understanding of these intricacies is essential for the development of effective control strategies of EP. In order to improve our knowledge about this interaction, laser-capture microdissection was used to collect bronchi, bronchi-associated lymphoid tissue, and lung parenchyma from animals infected with different strains of Mhyo, and mRNA expression levels of different molecules involved in Mhyo infection (ICAM1, IL-8, IL-10, IL-23, IFN-α, IFN-γ, TGF-ß, and TNF-α) were analyzed by qPCR. In addition, the quantification of Mhyo load in the different lung compartments and the scoring of macroscopic and microscopic lung lesions were also performed. Strain-associated differences in virulence were observed, as well as the presence of significant differences in expression levels of cytokines among lung compartments. IL-8 and IL-10 presented the highest upregulation, with limited differences between strains and lung compartments. IFN-α was strongly downregulated in BALT, implying a relevant role for this cytokine in the immunomodulation associated with Mhyo infections. IL-23 was also upregulated in all lung compartments, suggesting the potential involvement of a Th17-mediated immune response in Mhyo infections. Our findings highlight the relevance of Th1 and Th2 immune response in cases of EP, shedding light on the gene expression levels of key cytokines in the lung of pigs at a microscopic level.

Pathogens Associated with Bovine Mastitis: The Experience of Bosnia and Herzegovina.

To obtain improved insights into the complex microbial aetiology of bovine mastitis, this study investigated the pathogens involved in cattle mastitis in Bosnia and Herzegovina. A total of 179 milk samples from cows with clinical mastitis (CM) and subclinical mastitis (SCM), as well as eight bulk tank milk (BTM) samples from 48 dairy farms, were analysed by standard bacteriological and mycological methods. Mycoplasma detection and identification were performed using culture techniques and real-time polymerase chain reaction (PCR). A total of 88 (49.2%) mastitis samples were positive for known mastitis pathogens at 32 of 47 farms (68.1%). Mycoplasma bovis was a predominant pathogen (25/187; 13.4%) in the majority of herds (14/48; 29.2%) and accounted for 48.9% of positive CM samples. Escherichia coli was the second most dominant CM pathogen (34%), followed by Streptococcus agalactiae (10.6%), whereas Staphylococcus aureus and coagulase-negative staphylococci were the most common in SCM samples (17.1%). Other mastitis pathogens included Candida spp. and Prototheca zopfii. Two BTM samples were positive for M. bovis only, and one was positive for a mixed culture of S. aureus and Streptococcus uberis. The finding of various causative agents of bovine mastitis, with M. bovis emerging as the main pathogen, emphasizes the significance of comprehensive testing that includes not only common mastitis pathogens but also mycoplasmas, fungi, and algae.

Contagious Bovine Pleuropneumonia: A Passage to India.

The World Organization for Animal Health (WOAH)-listed contagious bovine pleuropneumonia (CBPP) emerged first in Europe and then spread to Eastern Asia, including Japan and China, from the Northern Territories of Australia at the end of the 19th century. Its route to India, however, is less well known as there is little evidence for large importations of cattle from Australia. The lack of accurate diagnostic tests at this time meant veterinary authorities relied solely on clinical and pathological signs, many of which were non-specific. Consequently, any diagnoses of CBPP reported in the early 20th century must be viewed with caution. More convincing reports of CBPP confirmed by laboratory tests were made in the 1930s and 1940s in the Indian state of Assam. Eradication campaigns began in the 1940s with immunizations of live attenuated vaccines and then more comprehensively in the 1950s and 1960s, supplemented with serological screening and the establishment of quarantine centres at international borders. The last case of CBPP, reported to WOAH, was seen in 1990, but the launch of a new awareness campaign in Assam in 2002 and recent reports of the disease in Pakistan suggests the disease has persisted in the Indian subcontinent well into the 21st century.

Mycoplasma ovipneumoniae: A Most Variable Pathogen.

Mycoplasma ovipneumoniae, a well-established respiratory pathogen of sheep and goats, has gained increased importance recently because of its detection in wild ruminants including members of the Cervidae family. Despite its frequent isolation from apparently healthy animals, it is responsible for outbreaks of severe respiratory disease which are often linked to infections with multiple heterologous strains. Furthermore, M. ovipneumoniae is characterized by an unusually wide host range, a high degree of phenotypic, biochemical, and genomic heterogeneity, and variable and limited growth in mycoplasma media. A number of mechanisms have been proposed for its pathogenicity, including the production of hydrogen peroxide, reactive oxygen species production, and toxins. It shows wide metabolic activity in vitro, being able to utilize substrates such as glucose, pyruvate, and isopropanol; these patterns can be used to differentiate strains. Treatment of infections in the field is complicated by large variations in the susceptibility of strains to antimicrobials, with many showing high minimum inhibitory concentrations. The lack of commercially available vaccines is probably due to the high cost of developing vaccines for diseases in small ruminants not presently seen as high priority. Multiple strains found in affected sheep and goats may also hamper the development of effective vaccines. This review summarizes the current knowledge and identifies gaps in research on M. ovipneumoniae, including its epidemiology in sheep and goats, pathology and clinical presentation, infection in wild ruminants, virulence factors, metabolism, comparative genomics, genotypic variability, phenotypic variability, evolutionary mechanisms, isolation and culture, detection and identification, antimicrobial susceptibility, variations in antimicrobial susceptibility profiles, vaccines, and control.

Vaccines for Mycoplasma Diseases of Small Ruminants: A Neglected Area of Research.

Mycoplasmas cause some of the most economically important diseases of sheep and goats, including diseases listed by the World Organisation for Animal Health (OIE) such as contagious caprine pleuropneumonia (CCPP) and contagious agalactia (CA). Other important mycoplasma diseases include chronic respiratory and arthritic syndrome (CRAS) and atypical pneumonia, both present on all continents where small ruminants are farmed. Unfortunately, owing to a lack of investment, most commercial vaccines for these diseases are of poor quality, being mostly composed of killed bacteriocins of dubious or unknown efficacy. Several Mediterranean laboratories produce autogenous vaccines, but these can only be used on farms where outbreaks have been officially declared, and consequently have limited impact on disease nationally. Effective live vaccines are available, but their use is often restricted because of safety concerns. With the necessary safeguards in place, we argue for their greater use. This review examines reported vaccines for mycoplasma diseases of small ruminants and attempts to identify new candidate antigens that may enable the development of improved products. Vaccines for CCPP are covered elsewhere.

Mycoplasma agalactiae: The Sole Cause of Classical Contagious Agalactia?

Contagious agalactia (CA) is suspected when small ruminants show all or several of the following clinical signs: mastitis, arthritis, keratoconjunctivitis and occasionally abortion. It is confirmed following mycoplasma isolation or detection. The historical and major cause is Mycoplasma agalactiae which was first isolated from sheep in 1923. Over the last thirty years, three other mycoplasmas (Mycoplasma mycoides subsp. capri, Mycoplasma capricolum subsp. capricolum and Mycoplasma putrefaciens) have been added to the etiology of CA because they can occasionally cause clinically similar outcomes though nearly always in goats. However, only M. agalactiae is subject to animal disease regulations nationally and internationally. Consequently, it makes little sense to list mycoplasmas other than M. agalactiae as causes of the OIE-listed CA when they are not officially reported by the veterinary authorities and unlikely to be so in the future. Indeed, encouraging countries just to report M. agalactiae may bring about a better understanding of the importance of CA. In conclusion, we recommend that CA should only be diagnosed and confirmed when M. agalactiae is detected either by isolation or molecular methods, and that the other three mycoplasmas be removed from the OIE Manual of Diagnostic Tests and Vaccines in Terrestrial Animals and associated sources.

Recent Developments in Vaccines for Bovine Mycoplasmoses Caused by Mycoplasma bovis and Mycoplasma mycoides subsp. mycoides.

Two of the most important diseases of cattle are caused by mycoplasmas. Mycoplasma bovis is a world-wide bovine pathogen that can cause pneumonia, mastitis and arthritis. It has now spread to most, if not all, cattle-rearing countries. Due to its increasing resistance to antimicrobial therapy, vaccination is the principal focus of the control of infection, but effective vaccines are currently lacking. Despite being eradicated from most parts of the world, Mycoplasma mycoides subsp. mycoides, the cause of contagious bovine pleuropneumonia (CBPP), continues to plague sub-Saharan Africa, affecting at least 25 countries. Numerous new experimental vaccines have been developed over the last 20 years to improve on protection afforded by the T1/44, a live vaccine in continuous use in Africa for over 60 years, but none so far have succeeded; indeed, many have exacerbated the disease. Tools for diagnosis and control are adequate for eradication but what is necessary are resources to improve vaccine coverage to levels last seen in the 1970s, when CBPP was restricted to a few countries in Africa. This paper summarizes the results of the main studies in the field of experimental mycoplasma vaccines, reviews data on commercially available bacterin vaccines and addresses issues relating to the search for new candidates for effective vaccines to reduce economic losses in the cattle industry caused by these two mycoplasmas.

Mycoplasma bovis Infections-Occurrence, Diagnosis and Control.

Mycoplasma bovis is a cause of bronchopneumonia, mastitis and arthritis but may also affect other main organs in cattle such us the eye, ear or brain. Despite its non-zoonotic character, M. bovis infections are responsible for substantial economic health and welfare problems worldwide. M. bovis has spread worldwide, including to countries for a long time considered free of the pathogen. Control of M. bovis infections is hampered by a lack of effective vaccines and treatments due to increasing trends in antimicrobial resistance. This review summarizes the latest data on the epizootic situation of M. bovis infections and new sources/routes of transmission of the infection, and discusses the progress in diagnostics. The review includes various recommendations and suggestions which could be applied to infection control programs.

The pathogen Mycoplasma dispar Shows High Minimum Inhibitory Concentrations for Antimicrobials Commonly Used for Bovine Respiratory Disease.

Mycoplasma dispar is an overlooked pathogen often involved in bovine respiratory disease (BRD), which affects cattle around the world. BRD results in lost production and high treatment and prevention costs. Additionally, chronic therapies with multiple antimicrobials may lead to antimicrobial resistance. Data on antimicrobial susceptibility to M. dispar is limited so minimum inhibitory concentrations (MIC) of a range of antimicrobials routinely used in BRD were evaluated using a broth microdilution technique for 41 M. dispar isolates collected in Italy between 2011-2019. While all isolates had low MIC values for florfenicol (<1 µg/mL), many showed high MIC values for erythromycin (MIC90 ≥8 µg/mL). Tilmicosin MIC values were higher (MIC50 = 32 µg/mL) than those for tylosin (MIC50 = 0.25 µg/mL). Seven isolates had high MIC values for lincomycin, tilmicosin and tylosin (≥32 µg/mL). More, alarmingly, results showed more than half the strains had high MICs for enrofloxacin, a member of the fluoroquinolone class considered critically important in human health. A time-dependent progressive drift of enrofloxacin MICs towards high-concentration values was observed, indicative of an on-going selection process among the isolates.

Infection Dynamics of Mycoplasma bovis and Other Respiratory Mycoplasmas in Newly Imported Bulls on Italian Fattening Farms.

Italian beef production is mainly based on a feedlot system where calves are housed with mixed aged cattle often in conditions favourable to bovine respiratory disease (BRD). In Veneto, an indoor system is also used for imported bulls around 300-350 kg. Mycoplasmas, in particular Mycoplasma bovis and Mycoplasma dispar, contribute to BRD in young calves, but their role in the disease in older cattle has not been investigated. In this study, ten heads of cattle were selected from each of the 24 groups kept in 13 different farms. Bulls were sampled by nasal swabbing at 0, 15, and 60 days after arrival for Mycoplasma isolation. Identification was carried out by 16S-rDNA PCR followed by denaturing gradient gel electrophoresis. M. bovis, M. dispar, and M. bovirhinis were identified, and prevalence was analysed by mixed-effects logistic regression models. This showed that most bulls arrived free of M. bovis, but within two weeks, approximately 40% became infected, decreasing to 13% by the last sampling. In contrast, the prevalence of M. dispar was not dependent on time or seasonality, while M. bovirhinis only showed a seasonality-dependent trend. The Italian fattening system creates an ideal environment for infection with M. bovis, probably originating from previously stabled animals.

The Implications of EU Regulation 2016/429 on Neglected Diseases of Small Ruminants including Contagious Agalactia with Particular Reference to Italy.

After almost 40 years, the 27 member states (MS) of the European Union (EU) will comply with the European Law 429/2016 in 2021 by completing a process of unification and harmonization of all regulations related to animal health between MS. These new provisions are based on modern scientific principles on animal health, on long-term epidemiological data, and, above all, on the most current risk assessment and analysis. The paper describes all changes and updates, which will impact the Italian current National regulation. A total of 58 animal diseases have been included in the Annex II ("Listing") and Annex IV ("Categorization") of the new Delegated Act (DA 2018/1629). Five diseases comprising the great viral epizooties were automatically included on the list because of their primary importance. These diseases include foot and mouth disease (FMD), African swine fever (ASF), classical swine fever (CSF), highly pathogenic avian influenza (HPAI), and African horse sickness (AHS). Another 53 diseases have been identified by the ad hoc assessment on listing and categorization of animal diseases developed by the European Food Safety Association. Seventeen communicable diseases of the Order Artiodactlya (sheep, goats, deer, etc.) have been listed including foot and mouth disease, sheep and goat pox, and pestes de petits ruminants. In addition, other endemic diseases affecting more than one species include blue tongue, tuberculosis, brucellosis, and anthrax. There are five categories (A-E) based on the degree of action to be undertaken throughout the EU for each disease. These vary from complete eradication for diseases not normally found in the EU like FMD (category A) for establishing surveillance for diseases like West Nile that present high risk but lack control tools (category E).

Validation of Loop-Mediated Isothermal Amplification (LAMP) Field Tool for Rapid and Sensitive Diagnosis of Contagious Agalactia in Small Ruminants.

Contagious agalactia (CA), an infectious disease of small ruminants, caused by Mycoplasma agalactiae, is responsible for severe losses to dairy sheep production with substantial socioeconomic impacts on small-scale farmers. The diagnosis of CA is still problematic, time-consuming and requires well-equipped labs for confirmation of outbreaks. Therefore, rapid, accurate and cost-effective diagnostic tests are urgently needed. This work aims to validate a novel Loop-Mediated Isothermal Amplification (LAMP) test, based on the p40 target gene, for the detection of M. agalactiae in dairy sheep in order to confirm its potential practical use as a rapid and cheap field test. The LAMP system proposed in this study consists of a portable device composed of real-time fluorometer with the automatic interpretation of results displayed in a tablet. A total of 110 milk samples (90 positives and 20 negatives) were analysed to optimise the analysis procedure and to investigate the efficacy and robustness of the LAMP method. All samples were analysed using LAMP and conventional real-time PCR to compare the diagnostic sensitivity of the methods. The sensitivity of the LAMP was 10-fold higher than that of real-time PCR, with a detection limit up to 103 CFU/ml. The LAMP assay was able to detect M. agalactiae in 81 of 90 (90%, 95%CI 0.84-0.96) positive milk samples compared to 69 (77%, 95%CI 0.59-0.95) positive samples detected by real-time PCR; no positive signal occurred for any of the negative milk samples in either test. Therefore, the LAMP assay was found to be more sensitive than real-time PCR, low-cost, easy to perform, fast and not affected by contamination, indicating its potential as an effective diagnostic tool in the field level for the diagnosis of CA.

Mycoplasma capricolum subsp. capripneumoniae, the cause of contagious caprine pleuropneumonia, comprises two distinct biochemical groups.

Mycoplasma capricolum subsp. capripneumoniae, the cause of the World Organisation of Animal Health- listed contagious caprine pleuropneumonia, is a member of the Mycoplasma mycoides cluster which comprises five pathogenic mycoplasmas of ruminants. These mycoplasmas are closely related immunologically and genetically which can lead to difficulties for differential diagnosis. The patterns of substrate metabolism of strains of M. c. capripneumoniae, gathered from diverse geographic regions, were studied by measurement of oxygen uptake rates. The strains fell into two major biochemical groups: one which only oxidised organic acids and glycerol and the other which could additionally metabolise sugars. Furthermore when DNA-DNA hybridisation tests were carried out these two groups of strains could be separated by their degree of DNA homology, the mean hybridisation value between members of the two groups was 86% well above the value of 70% normally used to indicate separate species. DNA-DNA hybridisation was also carried out between M. c. capripneumoniae strains and other members of the M. mycoides cluster. These experiments used labelled DNA from two representative subsp. capripneumoniae strains; these were 7/1a (organic acid-oxidising) and 4/2 LC (glucose-oxidising). The results showed a particularly close relationship of the glucose-oxidising strain to M leachii strains.

Mycoplasma tullyi sp. nov., isolated from penguins of the genus Spheniscus.

A mycoplasma isolated from the liver of a dead Humboldt penguin (Spheniscus humboldti) and designated strain 56A97T, was investigated to determine its taxonomic status. Complete 16S rRNA gene sequence analysis indicated that the organism was most closely related to Mycoplasma gallisepticum and Mycoplasma imitans(99.7 and 99.9 % similarity, respectively). The average DNA-DNA hybridization values between strain 56A97T and M. gallisepticum and M. imitans were 39.5 and 30 %, respectively and the Genome to Genome Distance Calculator gave results of 29.10 and 23.50 %, respectively. The 16S-23S rRNA intergenic spacer was 72-73 % similar to M. gallisepticum strains and 52.2 % to M. imitans. A partial sequence of rpoB was 91.1-92 % similar to M. gallisepticum strains and 84.7 % to M. imitans. Colonies possessed a typical fried-egg appearance and electron micrographs revealed the lack of a cell wall and a nearly spherical morphology, with an electron-dense tip-like structure on some flask-shaped cells. The isolate required sterol for growth, fermented glucose, adsorbed and haemolysed erythrocytes, but did not hydrolyse arginine or urea. The strain was compared serologically against 110 previously described Mycoplasma reference strains, showing that, except for M. gallisepticum, strain 56A97T is not related to any of the previously described species, although weak cross-reactions were evident. Genomic information, serological reactions and phenotypic properties demonstrate that this organism represents a novel species of the genus Mycoplasma, for which the name Mycoplasma tullyi sp. nov. is proposed; the type strain is 56A97T (ATCC BAA-1432T, DSM 21909T, NCTC 11747T).

Mycoplasmas: Brain invaders?

Mycoplasmas of humans and animals are usually associated with respiratory, autoimmune, genital and joint diseases. Human mycoplasmas have also been known to affect the brain. Severe central nervous system (CNS) diseases, such as encephalitis, have been linked to Mycoplasma pneumoniae and ureaplasma infections. Less well known is the sheep and goat pathogen, Mycoplasma agalactiae, which has been found in large quantities in the brain where it may be responsible for non-purulent encephalitis as well as ataxia in young animals. Experimental intra-mammary infections of sheep with this mycoplasma have resulted in histopathological changes in the CNS. The cattle pathogen, M. bovis, has been reported occasionally in the brains of calves and adult cattle showing a range of histopathological lesions including abscesses and fibrinous meningitis. Two avian pathogens, M. gallisepticum and M. synoviae have been isolated from the brains of poultry showing meningeal vasculitis and encephalitis. There have been no reported detections of two other avian pathogens, M. meleagridis or M. iowae in the CNS. Over the last few decades, mycoplasmas have been isolated from the brains of sea mammals dying in large numbers in the North Sea although it was concluded that their role may be secondary to underlying viral disease. Finally, evidence has been advanced that certain Spiroplasma species may have a role in the development of the transmissible spongiform encephalopathies (TSE). Invasion of the brain by mycoplasmas may be as a result of direct entry following damage to the inner ear as seen with M. bovis or across the blood brain barrier by mechanisms as yet uncertain.

Mycoplasma mastitis in cattle: To cull or not to cull.

Bovine mastitis caused by mycoplasmas, in particular Mycoplasma bovis, is a major problem for milk production and animal welfare in large dairy herds in the USA and a serious, although sporadic, disease in Europe and the Middle East. It causes severe damage to the udder of cattle and is largely untreatable by chemotherapy. Mycoplasma mastitis has a distinct epidemiology and a unique set of risk factors, the most important of which is large herd size. The disease is often self-limiting, disappearing within months of outbreaks, sometimes without deliberate intervention. Improved molecular diagnostic tests are leading to more rapid detection of mycoplasmas. Typing tests, such as multi-locus sequence typing, can help trace the source of outbreaks. An approach to successful control is proposed, which involves regular monitoring and rapid segregation or culling of infected cows. Serious consideration should be given by owners of healthy dairy herds to the purchase of M. bovis-free replacements. Increased cases of disease could occur in Europe and Israel if the trend for larger dairy herds continues.

Laboratory investigations into the origin of Mycoplasma synoviae isolated from a lesser flamingo (Phoeniconaias minor).

BACKGROUND: The role of wild birds in the transmission and spread of mycoplasmas is not clear. Up to now different Mycoplasma species have been isolated from wild birds many of which are not considered pathogens sensu stricto for domestic flocks. This report describes the first isolation of Mycoplasma synoviae in a captive lesser flamingo (Phoeniconaias minor) held in a zoo in Italy and the laboratory investigations performed to elucidate its origin. Results showed that the strain was similar to the MS-H vaccine strain using the vlhA methods although no vaccination with this product was used in the zoo. CASE PRESENTATION: This paper describes investigations into a case in which 10 of 12 adult lesser flamingos (Phoeniconaias minor) died after having recently been moved from the Netherlands to a new zoo in Northern Italy. While most of the birds appeared to have died from the stress of movement and poor adaptation to their new environment, Mycoplasma synoviae, an important poultry pathogen in the layer and meat industry, was isolated for the first time from the trachea of one animal presenting catarrhal tracheitis and fibrinous airsacculitis. Genetic analysis of the conserved region of the vlhA was not able to differentiate the flamingo strain from the MS-H vaccine strain. However differences in the sequences of the obg gene of the flamingo and vaccine strain were detected. A test for temperature-sensitivity (ts) gave a ts (-) phenotype for the flamingo strain, in contrast to the ts (+) status of the MS-H strain. Based on this information and knowing that the flamingos were not vaccinated against M. synoviae, it is highly likely that the flamingo was infected with a genetically similar wild strain by contact with infected birds. CONCLUSIONS: This case provides evidence for the potential role of international trade of ornamental birds as a possible route of introduction of new mycoplasma strains between countries, and moreover highlight that vlhA gene sequencing was not sufficient to discriminate the wild strain isolated from the flamingo from the MS-H vaccine strain.

Immunoproteomic characterisation of Mycoplasma mycoides subspecies capri by mass spectrometry analysis of two-dimensional electrophoresis spots and western blot.

OBJECTIVES: Mycoplasma mycoides subspecies capri is one of the causative agents of contagious agalactia in goats. The disease is characterised by mastitis, pneumonia, arthritis, keratitis and in acute cases septicaemia. No vaccine is currently available that has been demonstrated to prevent disease. METHODS: This study used two-dimensional electrophoresis to separate proteins from whole-cell preparations and tandem mass spectrometry to identify them. KEY FINDINGS: In total, 145 spots were successfully identified corresponding to 74 protein identities. Twenty of these proteins were found to be immunogenic by western blot analysis using a pooled serum sample from experimentally infected goats. CONCLUSIONS: Six proteins were found to have a less than 95% amino acid similarity to a closely related Mycoplasma species showing that they warrant further evaluation in development of diagnostic tests. These proteins were a dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex, phosphoglycerate kinase, pyrimidine-nucleoside phosphorylase, 30S ribosomal protein S6, ribulose-phosphate 3-epimerase and D-lactate dehydrogenase.